O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of …

An 18.5-kb human DNA segment was selected from a human XCharon-4A library by hybridization to mammalian valine tRNAiAc and found to encompass a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNAcuu gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region of the human DNA insert. At least nine Alu family members were found interspersed throughout the human DNA fragment. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase Ti fingerprints of the mature-sized tRNA transcription products are consistent with the DNA sequences of the structural genes. Three members of the chimpanzee triosephosphate isomerase (TPI) gene family, the functional transcription unit and two processed pseudogenes, were characterized by genomic blotting and DNA sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first complete hominoid TPI gene sequenced. The gene exhibits a very high identity with the human and rhesus TPI genes. In particular, the polypeptides of 248 amino acids encoded by the chimpanzee and human …

Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.

The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.

A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6- phosphogluconate) complexes. Initial velocity studies obtained as a function of pH and using NAD as the dinucleotide substrate for the reaction suggest that the 2'-phosphate is critical for productive binding of the dinucleotide substrate. Primary deuterium isotope effects using 3-<i-6-phosphogluconate were obtained for the 6-phosphogluconate dehydrogenase reaction using NADP and various alternative inucleotide substrates.

Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its …

Modification of proteins by ADP-ribose has been shown to be a versatile modification with respect to the amino acid side chain. The results described here will allow the study of the biological importance of ADP-ribose glycation and also allow differentiation on crude extracts between enzymatic modifications from protein ADP-ribose glycation that can occur due to the presence of NAD glycohydrolases.

This study demonstrates the presence of protein kinase C activity in both cytosolic and membrane fractions of bovine lens epithelial cells in culture. Protein kinase C activity is similar in normal and hyperglycemic cells. Furthermore, the ability of the enzyme to translocate from the cytosol to the membrane following phorbol ester treatment is unimpeded by hyperglycemic conditions. Moreover, protein kinase C activation had no effect on myoinositol uptake either in normal cells or in cells exposed to hyperglycemic conditions.

ADP-ribose polymer metabolism has been studied in human cells derived from a patient with Glutamyl Ribose Phosphate Storage Disease (GRPSD) and in mouse C3H1OT1/2 cells following oxidative stress induced by hydrogen peroxide (H202 ). It has been postulated that GRPSD resulted from an abnormality in ADP-ribose polymer metabolism. This study has shown that these cells exhibit reduced poly(ADP ribose) polymerase activity which is proposed to result from modification of the enzyme with ribose phosphate groups. The modification in the polymerase is proposed to be secondary to a defect in either ADP-ribosyl proteinlyase or an overproduction of a cellular phosphodiesterase. The metabolism of ADP-ribose polymers was rapidly altered by H202 and there were independent effects on adenine nucleotide pools. The results suggest that ADP-ribose polymer metabolism is involved in cellular defenses to oxidative stress.

O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.